TGF beta 1
Transforming growth factor beta type 1 (TGF beta-1) belongs to the superfamily of TGF beta proteins which includes bone morphogenic proteins (BMPs), avidins, inhibins, TGF beta proteins and several others. TGF beta signaling is involved in several cellular processes that include growth, develop ment, wound healing, cell differentiation and cell death (apoptosis) (1). TGF beta signaling has been implicated in diseases such as asthma, diabetes, Marfan Syndrome, cancer, Loeys-Dietz Syndrome and a few others. A variety of cell types secrete TGF beta in response to different stimuli. TGF beta is usually secreted as complex with the latency associated protein (LAP) and the latent TGF beta binding protein (LTGFBP). All three proteins are initially syntheiszed as a single polypeptide from the TGF beta gene before being proteolytically processed to yield the three different proteins. This complex prevents TGF-beta from being activated prematurely. Activation of mature TGF beta only occurs when specific proteinases such as plasmin release mature TGF beta from the latency complex in response to inflammation or other stimuli. TGF beta 1 signaling TGF beta 1 is the archetypical member of the TGF beta superfamily and it sign als through receptors that exist as heterodimers, TGF beta receptor type I and II). The ligand (TGF beta1) binds to the type II receptor which then recruit and phosphorylate the type I receptor to initiate a cascade of signaling events involving the phosphorylation of Smad proteins within the cell as depicted in the figure on the right. Signaling by TGF beta results in changes in gene expression which are responsible for the myriad effects of TGF beta. TGF beta signaling has been implicated in fibrotic diseases (7) as well as a number of cancer types. The recombinant TGF beta protein has been employed in the study of these diseases and developmental processes. In cancers however, TGF beta has opposing functions as a tumor supressor in the initial stages of the tumor and a tumor promoter in the later stages (2). The need to achieve a better understanding of how precancerous cells become transformed or how cancer cells become metastatic calls for the development of biological tools to facilitate research into those processes. Epithelial to mesenchymal transitions (EMT) which are important for organogeensis in the developing embryo as well as wound healing has been implicated in tumor development and metastasis. TGF beta family members have been shown to signal g ene expression changes involved in EMT and as such receombinant proteins of this family are being utilized to gain a better understanding of the microenvironment that leads to disease development when the process goes awry. In tumor progression, TGF beta is also believed to have tumor suppressive functions by inducing apoptosis and antagonizing tumor cell proliferation (3). Unfortunatley, TGF beta also signals for proliferation under conditions that are still being investigated and tumor cells are suspected to selectively enhance these properties of TGF beta while silencing its cytostatic and apoptotic functions to their own advantage. Other devastating fibrotic diseases that do not lead to cancer but compromise the quality of life of affected patients are also being investigated and recombinant TGF beta superfamily members are being used to test for possible antagonists and drug targets in the signaling pathway. rTGF beta 1 uses Recombinant human TGF beta 1 is added to cells in culture for research into the role of TGF beta signaling in angiogenesis, tumorigenesis, adaptive immunity, fibrosis, cell proliferation as well as developmental processes. rhTGF beta 1 construct/plasmid Recombinant human TGF-beta can be obtained as a lyophilized powder which has to be reconstituted for use in cell culture or in vivo experiments. In most cases, the expression system u sed is mammalian; namely, chinese hamster ovarian (CHO) cells are used and secreted TGF-beta is collected from the medium. Some constructs of TGF beta have been cloned into the pcDNA plasmid which has a glutamyl synthetase open reading frame for gene amplification into which the TGF beta gene is cloned (3). This was done to increase the yield of TGF beta recombinnant protein 10-fold. Additionally, some TGF-beta overexpression vectors are commercially available for transfecting your cell type of interest and some of these have bben cloned into the pCMV vector shown on the right. The His tag incorporated into the N-terminus of the protein after the secretion signal allows for pull down/easy purification of the protein after expression (2). A cysteine residue in the latent TGF beta binding protein (LTGFBP) product of the TGF beta gene is mutated to an alanine residue in the recombinant protein to prevent it from binding to the mature cytokine. By so doing, the secreted TGF beta is available as an active protein. To purify the mature protein from the culture supernantants, the supernantants are passed over Ni-NTA columns for affinity chromatography. By washing the columns with buffers containing excess imidazole at a low pH. The eluted protein is then incubated with a protease to cleave the his tag off and yeild the active protein in cases where the protein is availble commercially. For expression systems however, the His tag may not be followed by a cleavage signal if the experimental protocol the protein will be used in involves pull down assays. References 1. Shi Y and Massague J. (2003) Mechanisms of TGF beta signaling from cell membrane to the nucleus. Cell 113:686-700 2. http://www.biomedcentral.com/1471-2105/8/S9/S1/figure/F3?highres=y 3.Padua D and Massagué J (2009) Roles of TGF beta in metastasis. Cell Research 19:89-102 (pdf) 4. http://www.sinobiological.com/TGF-beta-and-Cancer-a-5898.html 5. http://www.creativebiomart.net/description_3904_9.htm 6. http://en.wikipedia.org/wiki/Transforming_growth_factor_beta 7. Branton MH and Kopp JB (1999) TGF-beta and fibrosis. Microbes. Infect. 1(15):1349-65